Registration Dossier
Registration Dossier
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EC number: 603-923-2 | CAS number: 135590-91-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471/472, Ames test): negative in S. typhimurium strains TA98, TA100, TA 102, TA1535, TA1537 and TA1538 and E. coli strain WP2uvrA with and without metabolic activation
Cytogenicity in mammalian cells in vitro (OECD 473, Chromosome aberration test): negative in Chinese hamster lung fibroblasts (V79 cells) with and without metabolic activation
Cytogenicity in mammalian cells in vitro (OECD 487, Micronucleus test): negative in cultured peripheral human lymphocytes with and without metabolic activation
Gene mutation in mammalian cells in vitro (OECD 476, HGPRT test): negative in Chinese hamster lung fibroblasts (V79 cells) with and without metabolic activation
Unscheduled DNA synthesis in mammalian cells in vitro (similar to OECD 482): negative in human A549 alveolar epithelial cells with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: unscheduled DNA synthesis (UDS) in mammalian cells in vitro
- Target gene:
- not applicable
- Species / strain / cell type:
- mammalian cell line, other: human A549 alveolar epithelial cell line
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MED + 10 % FCS, switched to arginine-deficient medium (MEM) with 10 mM hydroxyurea + 2 % FCS 2 days before start of treatment
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- Experiment 1 (± S9): 0.1, 0.3, 1, 2, 3, 10, 30, 100 µg/ml;
Experiment 2 (± S9): 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- benzo(a)pyrene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 3 h (exposure time)
- Fixation time (start of exposure up to fixation or harvest of cells): 3 h
NUMBER OF REPLICATIONS: 6 - Evaluation criteria:
- Statistically significant increase and dose dependent effect.
- Statistics:
- Data were statistically evaluated using Student's t-test.
- Species / strain:
- mammalian cell line, other: human A549 alveolar epithelial cell line
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Slight cytotoxic effect a concentration of 30-100 µg/ml without metabolic activation.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were performed to determine the solubility of the test
compound in the cell culture medium and to define the limit of visible microscopic
cytotoxocity.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test compound appeared soluble in the culture medium up to 1000 mg/ml. A slight cytotoxic effect was seen at a concentration of 30-100 µg/ml
without metabolic activation. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His-operon, Trp-operon
- Species / strain / cell type:
- S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 1. experiment: 0, 4, 20, 100, 500, 2500, 10000 µg/plate ± S9
2. experiment: 0, 4, 20, 100, 500, 2500, 5000 µg/plate ± S9 - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: N-Methyl-N-nitro-N-nitrosoguanidine (MNNG, -S9); 2.5 µg/plate for WP2uvra; 2-aminoanthracen (2-AA, +S9): 0.5 µg/plate for TA98, TA100 and TA1538, 1 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
NUMBER OF REPLICATIONS: tripliccates, 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of background lawn, reduction of colony count - Evaluation criteria:
- Significant increase in the number of revertant colonies, dose-dependency of effect
- Key result
- Species / strain:
- S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- starting at 2500 µg/plate; visible precipitation at 2500 microgram/plate only in the experiments without S9 (only buffer)
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the tested conditions, the test compound was not mutagenic in any of the five tested S. typhimurium strains (TA 98, TA 100, TA 1535, TA1537 and TA 1538) or in E. coli strain WP2 uvrA with and without metabolic activation up to 10000 µg/plate.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM + 10 % FCS
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- 18 h: 5.0, 12.5, 25.0 µg/ml (-S9); 10, 50, 100 µg/ml (+S9)
7 and 28 h: 25 µg/ml (-S9); 100 µg/ml (+S9) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3, 14 and 24 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 7, 18 and 28 hours, respectively
SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 2 % orcein solution
NUMBER OF REPLICATIONS: duplicate slides per group
NUMBER OF CELLS EVALUATED: 200 metaphases per experimental group
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, other: plating efficiency. The highest concentration did not reduce the number of scorable metaphases by more than 20 % of the negative controls. The mitotic index was determined in samples of 1000 cells. The toxicity of the test substance was determined in a preliminary experiment by establishing the concentration-related plating efficiency.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- The test substance is classified as mutagenic if:
- it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested. The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (binomial statistic with Fisher's exact test).
- there is a reproducible concentration related increase in the aberration rate.
The test substance is classified as non mutagenic when it tests negatively both with and without metabolic activation. - Statistics:
- Binomial statistic with Fisher's exact test.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In a preliminary experiment Hoe 107892 - substance technical was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 100 µg/ml. Following treatment in the absence of S9-mix, high cytotoxicity was observed at a concentration of 100 µg/ml. Survival declined in a dose-related manner reaching 1.8 % of the solvent control value at the highest dose level. In the presence of metabolic activation there was no indication of cytotoxicity up to 100 µg/ml. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HGPRT
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9-mix
- Test concentrations with justification for top dose:
- without S9-mix: 10, 25, 50, 75 and 100 µg/ml
with S9-mix: 25, 50, 75 and 100 µg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9,10-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: treatment of single culture at each dose level, divided into 5 subcultures for mutant selection; two independent experiments were performed
DETERMINATION OF CYTOTOXICITY
- Method: other: plating efficiency - Evaluation criteria:
- The test substance is classified as mutagenic if:
- it reproducibly induces with one of the test substance concentrations a mutation frequency that is statistically higher than the spontaneous mutant frequency in this experiment. (Biometry is to perform by the help of a statistic programm).
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data. - Statistics:
- Biometry was not necessary to perform.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
In a preliminary experiment Hoe 107892 - substance, technical was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 100 µg/ml.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The cytotoxicity experiment proved Hoe 107892 - substance, technical was not toxic to the V79 cells with S9-mix. Without S9-mix there was an indication of cytotoxicity at the highest concentration 100 µg/ml.
Based on these results 100 µg/ml was determined in the presence and in the absence of S9-mix as maximum dose level for mutagenicity testing in the main assays. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 Jul - 25 Jul 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 Jul 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted 30 May 2008
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary Experiment / Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate, with and without metabolic activation
Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate, with and without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The vehicle was chosen because of its solubility properties and its relative nontoxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenyl-diamine (4-NOPD) (-S9) 10 µg/plate in DMSO for TA98 and 50 µg/plate in DMSO for TA1537; 2-aminoanthracene (2-AA) (+S9) 2.5 µg/plate in DMSO for TA 1535, TA 1537, TA 98 and TA100, 10.0 µg/plate in DMSO for TA 102
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation)
Experiment 2: pre-incubation
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates in each experiment
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn - Evaluation criteria:
- The test material is considered mutagen in this test system if the following criteria are met:
- a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
- a dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration
- an increase at only one concentration is observed, which is considered biologically relevant if reproduced in an independent second experiment
- a dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Key result
- Species / strain:
- S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment 1 in the absence of metabolic activation and at 5000 µg/plate in experiment 2 with and without S9 mix. The undissolved particles had no influence on the data recording.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level.
HISTORICAL CONTROL DATA:
Please refer to “Any other information on results incl. tables”, Table No. 3 - Conclusions:
- Under the tested conditions, the test compound was not mutagenic in any of the five tested Salmonella typhimurium strains (TA 98, TA 100, TA 1535, TA1537 and TA 102) with and without metabolic activation up to 5000 µg/plate.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 Feb - 13 Jun 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 28 Jul 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 30 May 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- adopted August 1998
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Guideline Kanpoan No. 287 Environment Protection Agency "Eisei No. 127 - Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry"
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, Technical University Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Modal number of chromosomes: 22
- Karyotype: stable
MEDIA USED
- Type and identity of media: MEM containing Hank's salts supplemented with 10% FBS (except during 4 h treatment), neomycin (5 µg/mL) and amphotericin B (1%). Cultures were maintained at 37°C in a humidified atmosphere of 1.5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes (by treatment with HAT medium) - Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital / β-naphthoflavone
- Test concentrations with justification for top dose:
- Experiment 1:
Without S9 mix: 1.0, 2.0*, 4.0*, 8.0*, 16.0*, 32.0* and 64.0 µg/mL (4 h)
With S9 mix: 8.0*, 16.0*, 32.0*, 64.0*, 128.0* and 256.0 µg/mL (4 h)
Experiment 2:
Without S9 mix: 8.0*, 16.0*, 32.0*, 64.0*, 96.0* and 128.0 µg/mL (24 h)
With S9 mix: 16.0, 32.0*, 64.0*, 128.0*, 256.0* and 512.0* µg/mL (4 h)
* selected for analysis
The dose range of the first main experiment was set according to data generated in a pre-experiment, where cells were exposed for 4 h to test item concentrations in the range of 16.1 to 2058 µg/mL (equal to approx. 2 mg/mL of pure substance) in the presence and absence of S9 mix. Phase separation was observed at ≥ 128.6 µg/mL in all experiments. In the pre-experiment relevant cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed at 128.6 µg/mL and above without metabolic activation following 4h and 24h of treatment. In the presence of metabolic activation no relevant cytotoxic effect was determined up to the highest concentration.The dose range of the second main experiment was based on data generated in the pre-experiment (24 h treatment without metabolic activation) and in the first main experiment (4 h treatment with metabolic activation). - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (final concentration 0.5% (v/v))
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.7 to 1.2 × 10E7 cells (4 h treatment) and 0.2 × 10E7 cells (24 h treatment)
DURATION
- Exposure duration:
Experiment 1: 4 h exposure with and without S9 mix.
Experiment 2: 4 h exposure with S9 mix and 24 h without S9 mix.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
SELECTION AGENT (mutation assays): 11 µg/mL 6-thioguanine (6-TG)
NUMBER OF REPLICATIONS: duplicates each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency - Evaluation criteria:
- A test item is considered positive for gene mutation in this system if the following criteria were met:
- a concentration-related increase of the mutant frequency is observed exceeding the historical solvent control range
A test item is considered negative for gene mutation in this system if the following criteria were met:
- there is no concentration-related increase of the mutant frequency observed above the historical control range
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
A t-test was performed to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at ≥ 32.0 µg/mL without S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test substance.
- Precipitation: Precipitation visible at the end of treatment was noted in experiment 1 (4 h treatment) at 64.0 µg/mL in the absence of metabolic activation. Turbidity was observed in experiment 1 at ≥ 128.0 µg/mL in the presence of metabolic activation. In experiment 2 precipitation was noted after 4 h of treatment at ≥ 256 µg/mL in the presence of metabolic activation.
RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed in order to determine the toxicity of the test substance. The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test substance concentrations between 16.1 µg/mL and 2058 µg/mL were used. Cytotoxic effects were observed at 128.6 µg/mL and above without metabolic activation following 4 h and 24 h of treatment. In the presence of metabolic activation, no relevant cytotoxic effect was determined up to the highest concentration.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Phase separation occurred at 128.6 µg/mL and above in all experimental parts of the pre-experiment. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
HISTORICAL CONTROL DATA (Number of mutant colonies per 10E6 cells)
- Positive historical control data:
EMS: 150 and 225 µg/mL (4 h, -S9): Range: 53.9 – 889.0; mean value: 153.0 ± 88.5
EMS: 150 and 225 µg/mL (24 h, -S9): Range: 108.5 – 786.1; mean value: 341.6 ± 125.5
DMBA: 1.1 and 2.2 µg/mL (4 h, +S9): Range: 59.6 – 2024.6; mean value: 424.6 ± 291.4
- Negative (solvent/vehicle) historical control data: medium, acetone, water, DMSO, EtOH, THF
4 h, -S9: Range: 1.6 – 42.8; mean value: 15.0 ± 7.4; 95% confidence interval: 0.2 – 29.7
24 h, -S9: Range: 2.4 – 41.8; mean value: 14.2 ± 6.8; 95% confidence interval: 0.6 – 27.8
4 h, +S9: Range: 2.4 – 44.2; mean value: 14.6 ± 7.0; 95% confidence interval: 0.6 – 28.7
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by an adjusted cloning efficiency I below 50% in both cultures occurred at 32.0 µg/mL and above in the second experiment without metabolic activation (24 h treatment). The maximum concentration of the first and the second experiment without metabolic activation was not analysable due to exceedingly severe cytotoxicity. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 Mar - 09 Jun 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- adopted 26 Sep 2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- primary culture
- Details on mammalian cell type (if applicable):
- CELLS USED
- Cell proliferation: Blood samples were drawn from healthy non-smoking donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at low level. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Sex, age and number of blood donors if applicable:
- Donor for experiment IA: 25 years old male
- Donor for experiment IB: 29 years old female
- Donor for experiment IIA: 21 years old male
- Donor for experiment IIB: 33 years old female
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.
MEDIA USED
- Type and identity of media: DMEM/Ham’s F12, mixture (1:1) supplemented with 200 mM GlutaMAX, 100 U/mL / 100 µg/mL penicillin / streptomycin, 3 µg/mL of the mitogen phytohaemagglutinine (PHA), 10% FBS, 10 mM HEPEPS and 125 U.S.P.-U/mL heparin. During exposure to the test substance (4 h treatment), medium was used without FBS supplementation. Cultures were maintained at 37°C in a humidified atmosphere of 5.5% CO2 in air.
- Properly maintained: yes - Cytokinesis block (if used):
- Cytochalasin B (4 µg/mL)
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital / ß-naphthoflavone
- Test concentrations with justification for top dose:
- Preliminary experiment / Experiment IA:
4 h treatment (with metabolic activation, cells from donor IA): 4.37, 7.64, 13.4, 23.4, 40.9, 71.7*, 125*, 219*, 384, 672, 1176 and 2058 µg/mL
4 h treatment (without metabolic activation, cells from donor IB): 6.55, 11.5, 20.1*, 35.1*, 61.4*, 108, 188, 329, 823 and 2058 µg/mL
Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with the same top dose due to a technical error in Experiment IA, which was invalidated. The repeat test was designated Experiment IB.
Experiment IB:
4 h treatment (without metabolic activation, cells from donor IB): 6.55, 11.5, 20.1*, 35.1*, 61.4*, 108, 188, 329, 823 and 2058 µg/mL
Experiment II:
4 h treatment (with metabolic activation, cells from donor IIB): 23.4, 35.1, 52.7, 79.0, 119*, 178*, 267* and 400 µg/mL
20 h treatment (without metabolic activation, cells from donor IIA): 26.9, 35.0, 45.5*, 59.2*, 76.9*, 100, 200 and 400 µg/mL
* selected for analysis - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (final concentration 0.5% (v/v))
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Demecolcin (20 h, -S9), 125.0 ng/mL in water
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h
ACTIN POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B, 4 µg/mL
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. For details please refer to “Any other information on material and methods incl. tables”.
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI) - Evaluation criteria:
- A test substance was considered positive (able to induce chromosome breaks and/or gain or loss) in the micronucleus test if, in all of the experimental conditions examined
- At least one of the test concentrations exhibited a statistically significant increase in the frequency of micronucleated cells when compared to the concurrent solvent control
- The increase was concentration-related in at least one experimental condition
- The results were outside the range of the laboratory historical solvent control data - Statistics:
- Statistical significance was confirmed by using the Chi-squared test (α < 0.05) for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
- Key result
- Species / strain:
- lymphocytes: cultured peripheral human lymphocytes
- Remarks:
- primary culture
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Exp. IA and IB (± S9) and Exp. IIB (-S9): no cytotoxicity up to the highest evaluated concentration, which showed phase separation; Exp. IIA (-S9); concentrations showing clear cytotoxic effects were not evaluated for cytogenetic damage
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No relevant influence on osmolality or pH value was observed.
- Precipitation: Phase separation of the test item in the culture medium was observed at the end of treatment in Experiment IA at ≥ 219 µg/mL + S9 mix, in Experiment IB at ≥ 61.4 µg/mL - S9 mix, in Experiment IIA at ≥ 100 µg/mL - S9 and in Experiment IIB at ≥ 267 µg/mL + S9.
RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Test substance concentrations ranging from 4.37 to 2058 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-experiment for toxicity, phase separation of the test substance was observed at the end of treatment at ≥ 71.7 µg/mL in the absence of S9 mix and at ≥ 219 µg/mL in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation in the presence of S9 mix, the preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with the same top dose due to a technical error (wrong fixation solution used) in Experiment IA, which was invalidated. The repeat test was designated Experiment IB. In Experiment IB, phase separation of the test item was observed at the end of treatment at ≥ 61.4 µg/mL.
HISTORICAL CONTROL DATA
- Positive historical control data:
MMC: 1.0 µg/mL (4 h, -S9): mean value: 11.66%; range: 4.15 – 24.00; 95% control limit: 1.48 – 21.85
Demecolcin: 125.0 µg/mL (20 h, -S9): mean value: 3.55%; range: 2.10 – 6.40; 95% control limit: 1.69 – 5.41
CPA: (4 h, +S9): mean value: 4.80%; range: 2.25 – 11.30; 95% control limit: 0.88 – 8.73
- Solvent historical control data:
Solvents* (4 h, -S9): mean value: 0.61%; Range: 0.15 – 1.25; 95% control limit: 0.02 – 1.15
Solvents* (20 h, -S9): mean value: 0.55%; range: 0.05 – 1.43; 95% control limit: 0.05 – 1.05
Solvents* (4 h, +S9): mean value: 0.64%; range: 0.15 – 1.35; 95% control limit: 0.08 – 1.20
* Data of several aqueous and organic solvents
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment IA (4 h, + S9 mix) and IB (4h, - S9 mix) and in Experiment IIB (4 h, + S9 mix), no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment IIA (20 h, -S9 mix) after continuous treatment, concentrations showing clear cytotoxic effects were not evaluated for cytogenetic damage. - Conclusions:
- Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in cultivated peripheral human lymphocytes with and without metabolic activation.
Referenceopen allclose all
Maximum thymidine incorporations (mean ± SD):
|
Control |
Test substance (µg/ml) |
||
|
-S9 |
+S9 |
-S9 |
+S9 |
Experiment 1 |
1582 ± 86 |
1502 ± 280 |
2018 ± 181* (0.1) |
1708 ± 160 (30.0) |
Experiment 2 |
1587 ± 97 |
1680 ± 84 |
1600 ± 141 (10.0) |
1497 ± 201 (0.03) |
*p<0.01
A small but statistically significant increase in thymidine incorporation was observed only in the first experiment in the absence of S9-mix at the dose level of 0.1 µg/ml. No similar effect was obtained in the second experiment and no dose dependent effect occured. The second experiment was performed under the same experimental conditions.
Therefore this increase in the lowest concentration tested was considered as not relevant for the evaluation of genotoxicity. A statistically significant induction of unscheduled DNA synthesis was observed with the positive control substances. It was concluded that Hoe 107892 - substance, technical is inactive in the UDS-test under the experimental conditions described.
Maximum number of revertants (means):
|
Control |
Test substance (µg/plate) |
||
Strain |
-S9 |
+S9 |
-S9 |
+S9 |
TA100 |
138 |
166 |
134 (5000) |
161 (500) |
TA1535 |
10 |
12 |
10 (100) |
11 (4) |
TA1537 |
7 |
9 |
8 (4) |
10 (500) |
TA1538 |
11 |
16 |
14 (4) |
17 (2500) |
TA98 |
20 |
26 |
22 (4) |
29 (20 |
WP2uvrA |
23 |
30 |
26 (100) |
27 (4) |
The second experiment was chosen for presentation, actually there are no significant differences between either experiment 1 or 2.
Conclusion:
Hoe 107892 was found to be non-mutagenic in the bacterial systems tested either with or without exogenous metabolic activation at the dose levels used.
Chromosomal aberrations in V79 cells, summary of results:
|
|
|
|
|
Percent aberrant cells |
||
Test group |
No. of cells analyzed |
Dose (µg/ml) |
S9-mix |
Fixation interval (h) |
Incl. gaps |
Excl. gaps |
Exchanges |
Solvent |
200 |
0.0 |
- |
7 |
2.0 |
1.0 |
0.0 |
Test article |
200 |
25.0 |
- |
7 |
3.5 |
1.5 |
0.5 |
Solvent |
200 |
0.0 |
+ |
7 |
3.0 |
2.5 |
0.5 |
Test article |
200 |
100.0 |
+ |
7 |
5.0 |
4.0 |
0.5 |
Negative |
200 |
0.0 |
- |
18 |
2.5 |
1.5 |
0.0 |
Solvent |
200 |
0.0 |
- |
18 |
2.5 |
1.0 |
0.0 |
Positive |
100 |
2000.0 |
- |
18 |
33.0 |
33.0 |
25.0 |
Test article |
200 |
5.0 |
- |
18 |
0.5 |
0.5 |
0.5 |
Test article |
200 |
12.5 |
- |
18 |
1.5 |
1.0 |
0.5 |
Test article |
200 |
25.0 |
- |
18 |
2.0 |
1.5 |
0.5 |
Negative |
200 |
0.0 |
+ |
18 |
0.5 |
0.0 |
0.0 |
Solvent |
200 |
0.0 |
+ |
18 |
0.5 |
0.0 |
0.0 |
Positive CPA |
100 |
5.0 |
+ |
18 |
37.0 |
32.0 |
15.0 |
Test article |
200 |
10.0 |
+ |
18 |
1.5 |
1.5 |
0.0 |
Test article |
200 |
50.0 |
+ |
18 |
2.0 |
2.0 |
0.5 |
Test article |
200 |
100.0 |
+ |
18 |
2.0 |
1.5 |
1.0 |
Solvent |
200 |
0.0 |
- |
28 |
1.0 |
1.0 |
0.5 |
Test article |
200 |
25.0 |
- |
28 |
2.0 |
1.5 |
0.5 |
Solvent |
200 |
0.0 |
+ |
28 |
2.0 |
1.5 |
0.5 |
Test article |
200 |
100.0 |
+ |
28 |
1.5 |
1.5 |
0.5 |
Conclusion:
Hoe 107892 does not induce chromosome mutations (aberrations) in V79 Chinese hamster cells either in the presence or in the absence of a metabolic activation system under the experimental conditions tested. Therefore, Hoe 107892 is considered to be non-mutagenic in this chromosome aberration assay.
Maximum mutation frequencies and corresponding numbers of mutant colonies (means ± SD):
-S9 |
+S9 |
||
No. of mutant colonies (dose per ml) |
Mutation frequency |
No. of mutant colonies |
Mutation frequency |
5.8 ± 1.64 (negative) |
23.2 |
2.2 ± 1.64 (negative) |
9.9 |
2.8 ± 1.92 (solvent) |
11.1 |
3.2 ± 1.79 (solvent) |
16.0 |
118.0 ± 16.61 (EMS 1.0 mg) |
492.9 |
100.4 ± 9.99 (DMBA 7.7 µg) |
517.3 |
7.4 ± 2.79 (10.0 µg) |
27.7 |
4.0 ± 2.00 (100.0 µg) |
22.2 |
No relevant reproducible enhancement of the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
The results lead to the conclusion that the test compound is not mutagenic in the HGPRT-test with cells of the V79 Chinese hamster cell line.
Table No. 1: Experimental results, Experiment 1 (Preliminary experiment)
| Metabolic Activation | Test group | Dose (µg/plate) | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 102 |
| without S9 | DMSO | 14 ± 6 | 9 ± 3 | 27 ± 5 | 111 ± 4 | 422 ± 20 | |
| Untreated | 15 ± 5 | 9 ± 2 | 34 ± 1 | 115 ± 1 | 385 ± 19 | ||
| Test item | 3 µg | 13 ± 3 | 10 ± 4 | 28 ± 6 | 104 ± 9 | 370 ± 19 | |
| 10 µg | 13 ± 3 | 13 ± 1 | 24 ± 2 | 107 ± 7 | 393 ± 22 | ||
| 33 µg | 16 ± 2 | 8 ± 1 | 25 ± 4 | 100 ± 6 | 404 ± 21 | ||
| 100 µg | 16 ± 4 | 11 ± 2 | 30 ± 8 | 110 ± 15 | 423 ± 17 | ||
| 333 µg | 14 ± 6 | 8 ± 2 | 25 ± 5 | 102 ± 7 | 341 ± 34 | ||
| 1000 µg | 17 ± 4 | 8 ± 2 | 22 ± 1 | 100 ± 7 | 367 ± 47 | ||
| 2500 µg | 15 ± 5P | 12 ± 2P | 23 ± 4P | 96 ± 10P | 405 ± 16P | ||
| 5000 µg | 12 ± 2P M | 9 ± 1P M | 27 ± 1P M | 95 ± 5P | 417 ± 24P | ||
| NaN3 | 10 µg | 2128 ± 50 | 2098 ± 31 | ||||
| 4-NOPD | 10 µg | 312 ± 18 | |||||
| 4-NOPD | 50 µg | 73 ± 4 | |||||
| MMS | 2.0 µL | 4893 ± 244 | |||||
| with S9 | DMSO | 16 ± 3 | 19 ± 4 | 40 ± 2 | 164 ± 20 | 545 ± 7 | |
| Untreated | 23 ± 6 | 21 ± 9 | 33 ± 10 | 158 ± 11 | 510 ± 65 | ||
| Test item | 3 µg | 20 ± 6 | 20 ± 5 | 46 ± 11 | 145 ± 20 | 494 ± 54 | |
| 10 µg | 16 ± 1 | 19 ± 4 | 36 ± 8 | 155 ± 4 | 548 ± 35 | ||
| 33 µg | 16 ± 1 | 18 ± 2 | 45 ± 5 | 168 ± 7 | 598 ± 21 | ||
| 100 µg | 17 ± 4 | 16 ± 4 | 39 ± 5 | 119 ± 4 | 594 ± 12 | ||
| 333 µg | 14 ± 4 | 17 ± 8 | 48 ± 5 | 99 ± 13 | 454 ± 47 | ||
| 1000 µg | 17 ± 4 | 19 ± 3 | 40 ± 8 | 107 ± 11 | 489 ± 22 | ||
| 2500 µg | 17 ± 2 | 21 ± 7 | 37 ± 9 | 104 ± 12 | 478 ± 49 | ||
| 5000 µg | 17 ± 2 | 16 ± 3 | 40 ± 10 | 99 ± 14 | 459 ± 51 | ||
| 2-AA | 2.5 µg | 513 ± 37 | 521 ± 25 | 3598 ± 151 | 4379 ± 315 | ||
| 2-AA | 10.0 µg | 2892 ± 111 | |||||
| NaN3: sodium azide, 2-AA: 2-aminoanthracene; MMS: methyl methane sulfonate; 4-NOPD: 4-nitro-o-phenylene-diamine; P: precipitation; M: manual count | |||||||
Table No. 2: Experimental results, Experiment 2
| Metabolic Activation | Test group | Dose (µg/plate) | TA 1535 | TA 1537 | TA 98 | TA 100 | TA 102 |
| without S9 | DMSO | 16 ± 1 | 12 ± 6 | 24 ± 3 | 90 ± 3B M | 335 ± 15 | |
| Untreated | 17 ± 3 | 11 ± 3 | 27 ± 8 | 95 ± 9B M | 338 ± 12 | ||
| Mefenpyr- | 33 µg | 18 ± 3 | 9 ± 3 | 26 ± 5 | 89 ± 8B M | 355 ± 30 | |
| diethyl | 100 µg | 18 ± 4 | 10 ± 3 | 21 ± 4 | 90 ± 16B M | 361 ± 1 | |
| 333 µg | 20 ± 1 | 13 ± 1 | 24 ± 3 | 93 ± 13B M | 359 ± 10 | ||
| 1000 µg | 20 ± 1 | 11 ± 3 | 20 ± 1 | 91 ± 7B M | 315 ± 33 | ||
| 2500 µg | 15 ± 4 | 9 ± 4 | 17 ± 6 | 95 ± 6B M | 354 ± 5 | ||
| 5000 µg | 15 ± 5P | 9 ± 1P | 21 ± 3P | 83 ± 5P B M | 326 ± 6P | ||
| NaN3 | 10 µg | 2185 ± 103 | 1257 ± 97B M | ||||
| 4-NOPD | 10 µg | 386 ± 10 | |||||
| 4-NOPD | 50 µg | 81 ± 7 | |||||
| MMS | 2.0 µL | 2377 ± 53 | |||||
| with S9 | DMSO | 18 ± 4 | 21 ± 1 | 35 ± 8 | 117 ± 14B M | 449 ± 18 | |
| Untreated | 19 ± 4 | 25 ± 4 | 38 ± 4 | 123 ± 9B M | 526 ± 74 | ||
| Mefenpyr- | 33 µg | 18 ± 4 | 20 ± 1 | 40 ± 5 | 124 ± 10B M | 448 ± 12 | |
| diethyl | 100 µg | 15 ± 4 | 18 ± 5 | 39 ± 8 | 120 ± 12B M | 478 ± 55 | |
| 333 µg | 20 ± 6 | 18 ± 3 | 32 ± 3 | 118 ± 4B M | 586 ± 42 | ||
| 1000 µg | 14 ± 2 | 22 ± 4 | 35 ± 6 | 111 ± 15B M | 458 ± 74 | ||
| 2500 µg | 12 ± 5 | 20 ± 4 | 29 ± 3 | 112 ± 11B M | 417 ± 33 | ||
| 5000 µg | 15 ± 2P | 19 ± 5P | 34 ± 3P | 86 ± 11P B M | 306 ± 44P | ||
| 2-AA | 2.5 µg | 451 ± 26 | 400 ± 11 | 2748 ± 1000 | 1355 ± 200 BM | ||
| 2-AA | 10.0 µg | 2883 ± 185 | |||||
| NaN3: sodium azide, 2-AA: 2-aminoanthracene; MMS: methyl methane sulfonate; 4-NOPD: 4-nitro-o-phenylene-diamine; E: extensive bacterial growth; P: precipitation; M: manual count | |||||||
Table No. 3: Historical control data
| Strain | Control | without S9 mix | with S9 mix | ||||||
| Mean | SD | Min | Max | Mean | SD | Min | Max | ||
| TA 1535 | Solvent control | 14 | 2.37 | 9 | 23 | 20 | 3.75 | 11 | 35 |
| Untreated control | 14 | 2.87 | 7 | 25 | 20 | 3.82 | 10 | 32 | |
| Positive control | 1751 | 226.44 | 710 | 2385 | 367 | 93.12 | 126 | 703 | |
| TA1537 | Solvent control | 12 | 3.31 | 5 | 26 | 16 | 4.34 | 7 | 30 |
| Untreated control | 12 | 4.03 | 5 | 29 | 18 | 4.92 | 6 | 33 | |
| Positive control | 88 | 38.92 | 61 | 448 | 342 | 144.31 | 77 | 809 | |
| TA 98 | Solvent control | 30 | 5.6 | 17 | 47 | 40 | 6.08 | 21 | 58 |
| Untreated control | 31 | 6.36 | 17 | 55 | 42 | 6.83 | 24 | 68 | |
| Positive control | 372 | 78.05 | 158 | 595 | 2167 | 717.6 | 249 | 4089 | |
| TA 100 | Solvent control | 142 | 29.42 | 86 | 243 | 156 | 29.4 | 99 | 249 |
| Untreated control | 150 | 28.19 | 86 | 248 | 163 | 31.26 | 94 | 281 | |
| Positive control | 1741 | 488.75 | 569 | 3082 | 2642 | 796.59 | 825 | 4503 | |
| TA 102 | Solvent control | 380 | 43.64 | 305 | 510 | 502 | 89.88 | 321 | 677 |
| Untreated control | 372 | 41.71 | 300 | 479 | 511 | 87.27 | 315 | 659 | |
| Positive control | 2499 | 898.06 | 1080 | 4678 | 2329 | 598 | 1091 | 3972 | |
Mean: mean value of revertants/plate; SD: standard deviation; Min: minimal value; Max: maximal value
Table 1: Experimental results
| Concentration (µg/mL) | Precipitation | S9 mix | relative cloning efficiency I (%) | relative cell density (%) | relative cloning efficiency II (%) | mutant colonies / 10E6 cells | induction factor | relative cloning efficiency I (%) | relative cell density (%) | relative cloning efficiency II (%) | mutant colonies / 10E6 cells | induction factor | |
| Experiment 1 / 4 h treatment | culture I | culture II | |||||||||||
| Solvent control with DMSO | - | 100 | 100 | 100 | 17.1 | 0.2 - 29.7 | 100 | 100 | 100 | 20.1 | 0.2 - 29.7 | ||
| Positive control (EMS) | 300 | - | 128.3 | 62 | 79.5 | 180.4 | 0.2 - 29.7 | 86.7 | 62.3 | 54 | 235.2 | 0.2 - 29.7 | |
| Test item | 1 | - | 102.6 | 83.3 | 85.5 | 102.3 | 82.9 | 84.8 | # | ||||
| Test item | 2 | - | 93.6 | 65.8 | 61.6 | 10.5 | 0.2 - 29.7 | 94.6 | 90 | 85.2 | 22 | 0.2 - 29.7 | |
| Test item | 4 | - | 88.9 | 89.5 | 79.6 | 7.9 | 0.2 - 29.7 | 61.5 | 91.3 | 56.2 | 30.7 | 0.2 - 29.7 | |
| Test item | 8 | - | 120.9 | 61.5 | 74.4 | 22.6 | 0.2 - 29.7 | 78.6 | 69.8 | 54.8 | 27.7 | 0.2 - 29.7 | |
| Test item | 16 | - | 103.5 | 63.2 | 65.4 | 21.2 | 0.2 - 29.7 | 58.7 | 103.7 | 60.9 | 15.4 | 0.2 - 29.7 | |
| Test item | 32 | - | 113.4 | 42.7 | 48.5 | 10.7 | 0.2 - 29.7 | 74.8 | 85.2 | 63.7 | 17.6 | 0.2 - 29.7 | |
| Test item | 64 | P | - | culture was not continued## | 5.1 | 12.5 | 0.6 | ## | |||||
| Solvent control with DMSO | + | 100 | 100 | 100 | 13.9 | 0.6 - 28.7 | 100 | 100 | 100 | 27.7 | 0.6 - 28.7 | ||
| Positive control (DMBA) | 2.3 | + | 100.9 | 91 | 91.8 | 160.3 | 0.6 - 28.7 | 103.5 | 89.7 | 92.8 | 193.3 | 0.6 - 28.7 | |
| Test item | 8 | + | 100.5 | 93.9 | 94.4 | 17.2 | 0.6 - 28.7 | 98.7 | 88.9 | 87.7 | 18.1 | 0.6 - 28.7 | |
| Test item | 16 | + | 105.9 | 95.5 | 101.2 | 21.6 | 0.6 - 28.7 | 101.1 | 83.7 | 84.6 | 24.8 | 0.6 - 28.7 | |
| Test item | 32 | + | 99.1 | 101.2 | 100.3 | 16.2 | 0.6 - 28.7 | 103.2 | 91.3 | 94.2 | 27.4 | 0.6 - 28.7 | |
| Test item | 64 | + | 98.7 | 103.9 | 102.5 | 9.6 | 0.6 - 28.7 | 102.9 | 75.2 | 77.4 | 19.3 | 0.6 - 28.7 | |
| Test item | 128 | T | + | 101.8 | 83 | 84.5 | 19.4 | 0.6 - 28.7 | 102.1 | 79 | 80.7 | 15.1 | 0.6 - 28.7 |
| Test item | 256 | T | + | 96.8 | 85.5 | 82.8 | ## | 98.6 | 75.7 | 74.6 | ## | ||
| Experiment 2 / 24 h treatment | culture I | culture II | |||||||||||
| Solvent control with DMSO | - | 100 | 100 | 100 | 13.1 | 0.2 - 29.7 | 100 | 100 | 100 | 8.2 | 0.2 - 29.7 | ||
| Positive control (EMS) | 300 | - | 88.8 | 55.9 | 49.6 | 449.4 | 0.2 - 29.7 | 50.3 | 70.1 | 35.2 | 395.1 | 0.2 - 29.7 | |
| Test item | 8 | - | 103.6 | 56.2 | 58.2 | 13.8 | 0.2 - 29.7 | 72.1 | 83.1 | 59.9 | 13.4 | 0.2 - 29.7 | |
| Test item | 16 | - | 91.2 | 86.7 | 79.1 | 9.7 | 0.2 - 29.7 | 80.8 | 71.4 | 57.6 | 9 | 0.2 - 29.7 | |
| Test item | 32 | - | 64.2 | 57.3 | 36.8 | 9.1 | 0.2 - 29.7 | 70.2 | 43.7 | 30.7 | 6.1 | 0.2 - 29.7 | |
| Test item | 64 | - | 70.4 | 44.4 | 31.2 | 8.7 | 0.2 - 29.7 | 70.7 | 25.1 | 17.7 | 10.6 | 0.2 - 29.7 | |
| Test item | 96 | - | 0 | 31.4 | 0 | 10.5 | 0.2 - 29.7 | 0 | 31.7 | 0 | 6.7 | 0.2 - 29.7 | |
| Test item | 128 | - | 0 | 35.4 | 0 | ## | 0 | 22.5 | 0 | ## | |||
| Experiment 2 / 4 h treatment | culture I | culture II | |||||||||||
| Solvent control with DMSO | + | 100 | 100 | 100 | 7.6 | 0.6 - 28.7 | 100 | 100 | 100 | 17.1 | 0.6 - 28.7 | ||
| Positive control (DMBA) | 2.3 | + | 88.1 | 74 | 65.2 | 94.9 | 0.6 - 28.7 | 80.6 | 96 | 77.4 | 101.2 | 0.6 - 28.7 | |
| Test item | 16 | + | 84.9 | 108.6 | 92.1 | # | 94.9 | 93.1 | 88.4 | # | |||
| Test item | 32 | + | 92.5 | 97.3 | 90.1 | 14.4 | 0.6 - 28.7 | 97.2 | 96.4 | 93.7 | 11.9 | 0.6 - 28.7 | |
| Test item | 64 | + | 99.5 | 96.3 | 95.9 | 14.6 | 0.6 - 28.7 | 100.2 | 102.6 | 102.8 | 7.6 | 0.6 - 28.7 | |
| Test item | 128 | + | 91.6 | 98.6 | 90.3 | 18.5 | 0.6 - 28.7 | 105.7 | 101 | 106.8 | 12 | 0.6 - 28.7 | |
| Test item | 256 | P | + | 75.2 | 85.9 | 64.6 | 10.3 | 0.6 - 28.7 | 108.4 | 98.8 | 107.1 | 9.8 | 0.6 - 28.7 |
| Test item | 512 | P | + | 61.5 | 81.6 | 50.2 | 13.6 | 0.6 - 28.7 | 93.2 | 89.7 | 83.7 | 14.5 | 0.6 - 28.7 |
| T = Turbidity; P= Precipitation at the end of treatment # culture was not continued as a minimum of only four analysable concentrations is required ## culture was not continued due to exceedingly severe cytotoxic effects ### culture was not continued to avoid analysis of too many insoluble concentrations |
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Table 1: Experimental results of the micronucleus test
| Exp. | Preparation interval | Test item concentration (µg/mL) | Proliferation index (CBPI) | Cytostasis in %* | Micronucleated cells (in % **) |
| Exposure period 4 h without S9 mix | |||||
| IB | 40 h | DMSO 0.5% (v/v) | 2.03 | 0.15 | |
| Mitomycin C 1.0 µg/mL | 1.86 | 16.5 | 10.40S | ||
| 20.1 | 1.96 | 6.6 | 0.10 | ||
| 35.1 | 1.99 | 3.5 | 0.15 | ||
| 61.4PS | 1.89 | 14.1 | 0.10 | ||
| Exposure period 20 h without S9 mix | |||||
| IIA | 40 h | DMSO 0.5% (v/v) | 1.85 | 0.35 | |
| Demecolcin 125.0 ng/mL | 1.33 | 60.4 | 3.40S | ||
| 45.5 | 1.77 | 9.2 | 0.55 | ||
| 59.2 | 1.60 | 29.1 | 0.20 | ||
| 76.9 | 1.58 | 31.7 | 0.55 | ||
| Exposure period 4 h with S9 mix | |||||
| IA | 40 h | DMSO 0.5% (v/v) # | 1.85 | 1.13 | |
| Cyclophosphamide 17.5 µg/mL | 1.60 | 29.1 | 9.75S | ||
| 71.7 | 1.93 | n.c. | 0.95 | ||
| 125 | 1.91 | n.c. | 0.70 | ||
| 219PS | 1.89 | n.c. | 1.00 | ||
| IIB | 40 h | DMSO 0.5% (v/v) | 2.03 | 1.20 | |
| Cyclophosphamide 12.5 µg/mL | 1.81 | 21.1 | 5.10S | ||
| 119# | 1.92 | 10.6 | 1.70 | ||
| 178# | 2.00 | 3.2 | 1.80 | ||
| 267 # PS | 1.96 | 7.1 | 1.50 | ||
| * For the positive control groups and the test item treatment groups the values are related to the solvent controls ** The number of micronucleated cells was determined in a sample of 2000 binucleated cells # The number of micronucleated cells was determined in a sample of 4000 binucleated cells for proof of validity PS: Phase separation occurred at the end of treatment S: The number of micronucleated cells is statistically significantly higher than corresponding control values n.c.: Not calculated as the CBPI is equal or higher than the solvent control value |
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In Experiment IIB (4 h treatment, + S9 mix), all evaluated concentrations showed increases in micronucleated cells (1.70, 1.80 and 1.50%) above the historical control data (95% control limit: 0.08 – 1.20%). Since the values were not statistically significant, did not occur in a dose-related manner and were not reproducible between Experiments IA and IIB, the findings were considered not biologically relevant. In addition, the concurrent control value was at the upper limit of the historical control range, with an increase in micronucleated cells of 1.20%.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Cytogenicity in mammalian cells in vivo (OECD 474, Micronucleus test): negative
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: mean males = 29.7 g (27 - 35g), females = 24.8 g (20 - 29 g)
- Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: starch mucilage
- Concentration of test material in vehicle: 250 mg/ml (25 % w/v)
- Amount of vehicle (if gavage or dermal): The test compound was given in two equal parts of 10 ml/kg bw each within two hours - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test compound dilutions were prepared fresh each day. The test compound was mortared, 6250 mg Hoe 107892 - substance, technical were weighed in a beaker, mixed with starch mucilage, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed. - Duration of treatment / exposure:
- 2 oral applications of half of the dose within 2 h
- Frequency of treatment:
- once
- Post exposure period:
- 24, 48, 72 h
- Dose / conc.:
- 5 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide (Endoxan)
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw - Tissues and cell types examined:
- bone marrow
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
According to a preliminary study to test the acute toxicity, a dose of 5000 mg Hoe 107892 - substance, technical per kg bodyweight was the maximum applicable dose level.
DETAILS OF SLIDE PREPARATION:
The animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure:
- 5 minutes in methanol
- 3 minutes in May-Gruenwalds solution
- 2 minutes in May-Gruenwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
METHOD OF ANALYSIS:
1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group which they belonged to remained unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically. - Statistics:
- Comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase). The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: reduced spontaneous activity and uncoordinated gait
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Hoe 107892 - substance, technical was within the normal range of the negative control groups. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound
- Statistical evaluation: No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. - Conclusions:
- Interpretation of results: negative
Reference
All animals survived after application of 5000 mg Hoe 107892 - substance, technical per kg bodyweight. No signs of toxicity were observed.
Summarizing it can be stated that, under the conditions described, administration of Hoe 107892 - substance, technical did not lead to a substantial increase of micronucleated polychromatic erythrocytes. It is concluded that Hoe 107892 - substance, technical is not mutagenic in the micronucleus test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
There is data from eight tests available examining genetic toxicity in vitro, including two bacterial reverse mutation assays in Salmonella typhimurium and Escherichia coli strains, one chromosomal aberration test in mammalian cells, two gene mutation assays in mammalian cells, one micronucleus test and one unscheduled DNA synthesis test in mammalian cells. All of these tests were negative for genotoxicity. In addition, a mammalian erythrocyte micronucleus test was performed in vivo, which was negative as well.
In vitro gene mutation in bacteria
Two Ames tests are available, analysing the test items mutagenic potential in bacteria (Ames test) according to OECD guidelines 471 and 472 and in compliance with GLP. The first Ames test was conducted with S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and with the E. coli strain WP2 uvr A. The second test was performed with S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. Both tests were performed in the presence and absence of metabolic activation (S9 mix). For each test, two independent experiments were performed using concentrations up to 10000 µg/plate. Vehicle (DMSO) and positive controls were included in each experiment.
The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains in any experiment, neither in the presence nor absence of metabolic activation. Positive and vehicle controls were in the range of historical control data and confirmed the validity of the test system. Thus, based on the results of the present study and under the experimental conditions chosen, the test substance is negative for reverse mutation in bacteria with and without metabolic activation.
In vitro gene mutation in mammalian cells
The test item was further investigated for its mutagenic potential in mammalian cells in vitro according to OECD guideline 476 and in compliance with GLP. In two independent studies, gene mutations in the HPRT locus were analysed in Chinese hamster lung fibroblasts (V79 cells) in the presence and absence of metabolic activation (S9 mix). In both tests, two independent experiments were performed.
In the first test, the cells were exposed for 4 h to test item concentrations in the range of 10 to 100 µg/mL without S9 mix and for 25 to 100 µg/mL with S9 mix. In the second test, the exposure was performed for 4 h using concentrations of 1 to 64 µg/mL without S9 mix and 8 to 512 µg/mL with S9 mix. In addition, cells were exposed for 24 h to concentrations of 8 to 128 µg/mL without S9 mix. Appropriate vehicle (DMSO) and positive controls were included.
In none of the experiments, treatment with the test item did significantly induce the number of forward mutations at the HPRT locus of V79 cells at any concentration, neither in the presence, nor in the absence of metabolic activation. Solvent controls remained within the range of historical control data, while the positive compounds markedly increased mutation frequencies, thus demonstrating the sensitivity and validity of the test system. Based on these results and under the experimental conditions reported, the test item is considered to be non-mutagenic in the HPRT test.
In vitro cytogenicity in mammalian cells
The clastogenic activity of the test substance was investigated in Chines hamster lung fibroblasts (V79 cells) according to OECD guideline 473 and in compliance with GLP. In the first experiment, the cells were exposed for 18 h to test item concentrations in the range of 5 to 25 µg/mL in the absence and 10 to 100 µg/mL in the presence of metabolic activation. In the second experiment, the cells were exposed for 7 and 28 h to 25 µg/mL without S9 mix and to 100 µg/mL with S9 mix. Appropriate negative, vehicle and solvent controls were included. Per experimental group, a number of 200 metaphases were evaluated for structural chromosomal aberrations.
Under any tested condition, the test material did not induce any toxicologically significant increases in the frequency of cells with aberrations with or without metabolic activation. Negative, vehicle and positive controls confirmed the validity of the test. Based on these findings, the test item was considered negative for chromosomal aberration in V79 cells.
In addition, the potential of the test item to induce micronuclei in mammalian cells in vitro was analysed in a micronucleus test in cultured human peripheral lymphocytes. The test was conducted according to OECD guideline 487 and in compliance with GLP. A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cells were incubated for 4 h in the presence and absence of metabolic activation (S9 mix) at concentrations in the range of 4.37 to 2058 µg/mL. Since the results fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1. In a second experiment, cells were incubated for 4 h with S9 mix at concentrations of 23.4 to 400 µg/mL with S9 mix and for 24 h at concentrations of 26.9 to 400 µg/mL without S9 mix. The cells were prepared 40 h after the start of treatment. For each experimental condition, two parallel cultures (at least 2000 binucleated cells per condition) were evaluated for cytogenetic damage.
In both experiments at any concentration, treatment with the test item revealed no biologically relevant increase in the frequency of micronucleated cells, both, in the presence and absence of metabolic activation. Mutant frequencies of vehicle controls (DMSO) remained within the range of historical negative control data.
Concurrent positive controls (cyclophosphamide (+S9), mitomycin C and demecolcine (-S9)) induced a statistically significant increase in micronuclei frequencies when compared to vehicle controls and the response was within the range of historic positive control data. Under the experimental conditions of the study and when tested up to phase separating concentrations, the test item has no clastogenic or aneugenic potential.
In vitro unscheduled DNA synthesis
The test item was analysed for unscheduled DNA synthesis in vitro in a test conducted similar to OECD guideline 482 and in compliance with GLP. In two independent experiments, human A549 alveolar epithelial cells were exposed to the test item, vehicle and positive controls in the presence and absence of metabolic activation (S9 mix). The cells were exposed to test item concentrations in the range of 0.1 to 100 µg/mL (first experiment) and 0.01 to 100 µg/mL (second experiment). After 3 h of exposure in the presence of3H-thymidine, the incorporation of tritiated thymidine was determined.
Treatment with the test item did not increase unscheduled DNA synthesis at any dose level when compared to vehicle controls. Marked increases in UDS were observed upon treatment with the positive control compound, demonstrating the validity of the test system.
Based on the results of the present study, the test substance did not induce UDS in human A549 alveolar epithelial cells in vitro under the tested conditions.
In vivo cytogenicity in mammalian cells
The test item was analysed for micronucleus formation in erythrocytes of NMRI mice according to OECD guideline 474 and compliant with GLP. Within 2 h of administration, each of the 5 male and female mice received two doses of 2500 mg/kg bw test item (5000 mg/kg bw in total) by oral gavage. Vehicle and positive control groups were included. Animals were killed 24, 48 and 72 h after administration and bone marrow cells were isolated. A number of 1000 polychromatic erythrocytes and 1000 normochromatic erythrocytes for each animal were scored for the presence of micronuclei.
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. In addition, the ratio of polychromatic to normochromoatic erythrocytes remained unaffected by the test compound. Positive and vehicle controls demonstrated the sensitivity and validity of the test system. Based under the conditions of the test, the test item was considered to be negative for micronucleus formation in vivo.
Justification for classification or non-classification
The available data on genetic toxicity in vitro and in vivo do not meet the criteria for classification according to Regulation No. (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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